Mouse Cytokine-II MultiGene-12™ RT-PCR Profiling Kit Cat. No. PM
Contents
1.2. one RNA sample can be analyzed in quadruplicate and a strip can be removed after 15 20 25 and 30 cycles Why did I not see a PCR product where expected to see one RNA quality is the most critical factor for a successful MultiGene 12 RT PCR Profiling analysis Be sure to check the quality of the RNA before use The ratio of the absorbance readings at 260 to 280 nm should be at least 1 8 and a total RNA sample should yield two distinct bands by agarose gel electrophoresis representing the 28S and 18S ribosomal RNA molecules Those bands should have a 2 1 intensity ratio as well The relative expression I observe with MultiGene 12 does not agree with my GEArray or other microarray result An inability to achieve verification is not necessarily due to a flaw in the array experiment Arrays may simply not be able to represent a particular gene accurately However RT PCR methods are better able to accurately represent the expression level of genes Monitoring the expression level of a large number of genes by RT PCR is difficult usually only a few are performed ata time Array based methods have the power of examining many genes simultaneously The changes in the expression level of a few genes are then verified by RT PCR The MultiGene 12 RT PCR Profiling Kit provides you the tools to verify or refute array results If you have any other questions please call our Technical Support representatives at 1 888 503 3187 or 301 682 9200
3. the same MultiGene 12 Primer Strip Close the tubes with their caps Mix by gently flicking the bottom of each tube c Place the strip on ice while setting up the PCR program 94 C 5 min 30 cycles of 94 C 30 sec 50 C 30 sec and 72 C 45 sec The PCR cycle number should be optimized for each experiment Try using 30 cycles at first The number of cycles can be decreased to 15 or increased to 45 See Troubleshooting Guide Place the strips in the PCR block and run the program SuperArray MultiGene 12 RT PCR Profiling Kit User Manual Version 1 1 8 15 2003 8 4 Product Characterization a When the PCR is complete load 10 ul of each reaction into separate wells of a 1 agarose gel containing 0 5 ug ml ethidium bromide in 1X TAE NOTE No gel loading dye or buffer is required The ReactionReady PCR master mix 1000 already contains gel loading dye b Load an appropriate amount of 100 bp DNA Step Ladder Promega G695A in an adjacent lane c Electrophorese in 1X TAE at 80V for 40 min Note The gel dye runs at a size of 50 bp under these conditions d Capture an image of the gel on a UV Trans Illuminator using a CCD camera or on high speed film The individual product information sheets list the expected sizes of the PCR products 5 Image Acquisition and Data Acquisition and Analysis a Image Acquisition At this time we highly recommend using a CCD camera to capture the image of the gel because the Data A
4. 1 0 mg ml 2 RT Reaction This step must be performed using a kit or reagents NOT included with the MultiGene 12 RT PCR Profiling Kit USA Customers may order the ReactionReady First Strand cDNA Synthesis Kit Catalog Number C 01 and follow the protocol found in Appendix A USA Customers may also order the individual components listed in the Additional Materials Required section and follow the protocol found in Appendix B instead Customers outside the USA must order the individual components listed in the Additional Materials Required section and follow the protocol found in Appendix B The C 01 kit is available in some international territories and the protocol in Appendix A may be used Please contact your local distributor for availability Note In order to take advantage of the Housekeeping MultiGene 12 RT PCR Profiling Kits two separate RT Reactions must be performed for each RNA sample One reaction will be used for a pathway specific MultiGene 12 primer strip and the other will be used for the Housekeeping MultiGene 12 primer strip 3 MultiGene 12 PCR a PCR Cocktail Transfer each completed 20 ul RT Reaction into separate tubes of ReactionReady PCR master mix 1000 Add 300 ul of ddH20 Allow the lyophilized components to dissolve for a few minutes by vortexing gently b Dispense 25 ul of a single PCR Cocktail ReactionReady PCR master mix 1000 combined with RT Reaction to each of the 12 PCR tubes of
5. Mouse Cytokine Il MultiGene 12 RT PCR Profiling Kit Ssu perArray Cat No PM 008A AF Bioscience Corporation Description The Mouse Cytokine II MultiGene 12 RT PCR Profiling Kit is designed to monitor the expression levels of the cytokine genes listed below The regulation of cytokine expression plays a fundamental role in the development and function of the immune system Cytokines are secreted by immune cells and bind to their cognate receptors on the surface of another cell The ability to secrete particular cytokines gives a cell the ability to signal to particular cells or initiate or propagate certain cascades Each MultiGene 12 RT PCR Profiling Kit contains 8 identical strips of 12 PCR tubes Each PCR tube contains a pair of gene specific primers that is carefully designed and tested by SuperArray Bioscience The kit also includes 8 tubes of ReactionReady PCR master mix 1000 a lyophilized master mix for end point analysis by agarose gel electrophoresis Kit Contents Component Po Amount ReactionReady PCR master mix 1000 8 tubes each enough for 12 reactions MultiGene 12 Primer Strips 8 strips of 12 PCR tubes containing primers Gene List Tube Gene Name RT PCR Gene Symbol UniGene GenBank Product Accession Expected 6 interferon gamma 391mg Mm 529_JAKO89574___ Bo Mit S o a i Mi Mm 2326 __ NM_010798 T a growth taciti gfb1 Mm 9154 NM_011577 gfb3 gfb3 Mm 3992 NM_009368 14 Lymphotoxi
6. Profiling Kit products includes a limited nonexclusive license to use the kit components for research use only This license does not grant rights to use the kit components for reproduction of any primer pair mix to modify kit components for resale or to use MultiGene 12 RT PCR Profiling Kits to manufacture commercial products without written approval of SuperArray Bioscience Corporation No other license expressed implied or by estoppel is granted U S patents may cover certain isolated DNA sequences included in the MultiGene 12 RT PCR Profiling Kit Presently it is not clear under U S laws whether commercial users must obtain licenses from the owners of the rights to these U S patents before using MultiGene 12 RT PCR Profiling Kits SuperArray MultiGene 12 RT PCR Profiling Kit User Manual Version 1 1 8 15 2003 4 I BACKGROUND AND INTRODUCTION The advancement of nucleic acid array technology has made it possible to analyze the expression of multiple genes in a single experiment However changes in the expression of genes observed by array analysis must still be verified by an independent method such as Northern or RT PCR analysis Only one gene can be analyzed by Northern analysis at one time in any given experiment making it difficult to characterize gene expression in a high throughput manner Multiple RT PCR analyses can be performed at one time with the 96 well blocks built into most thermal cyclers However the design of specif
7. cquisition will be more straightforward and the resulting data will have a wider dynamic range A high speed photograph can be used however the image must be digitized by other means separate software must be used and the dynamic range will be smaller b Data Acquisition If a CCD camera was used to capture the image of the gel the software accompanying the instrument should be able to convert the image of the bands into data Follow the instructions for that software If relying on a high speed photograph of the gel digitize the image of the gel using a desktop scanner to obtain a TIFF file of the image To convert that image into data we then recommend using the software NIH IMAGE Available free from the NIH See the following web page and its links for more information http rsb info nih gov nih image Follow the instructions for that software c Data Analysis During Data Acquisition remember to use the software to determine an appropriate background value That is use the same rectangle or other shape used to surround the gel bands to surround a blank area of the gel and obtain data from that area Subtract that background value from the integrated intensity values of all other data points obtained from the gel bands Finally divide each of the background corrected values by the background corrected value for the GAPDH housekeeping gene These normalized values can then be compared between experimental conditions For exampl
8. ding on the placement of the primer relative to the relevant splice junctions The existence of alternative splicing products may not necessarily have been published in the literature By the same token a single band of the expected size from the provided primer set does not necessarily indicate a single splicing product The single band may simply represent the dominant or major splicing product Multiple spicing variants can also produce a single RT PCR band if the amplified fragment lies within a single exon Contamination of your RNA with genomic DNA may also cause the appearance of extra PCR bands Does the gel loading dye interfere with the PCR No We have carefully tested the effect of the inert ingredients in the ReactionReady PCR master mix 1000 on the ability to obtain a PCR product None of the added components adversely affects the reaction How much RNA should I use for MultiGene 12 analyses that is why is the range of RNA amounts recommended The amount of RNA used can depend on how much you are able to isolate and how may different experiments you need to perform with that sample Also lower abundance messages may require more input RNA in order to observe an RT PCR product The RT PCR can accommodate as little RNA as 1 0 ug and as much as 5 0 ug per MultiGene 12 Primer Strip of 12 tubes Another way to control RNA loading in a MultiGene 12 analysis involves performing replicate analyses using the same RNA sample b
9. e fold changes in gene expression can be calculated SuperArray MultiGene 12 RT PCR Profiling Kit User Manual Version 1 1 8 15 2003 Appendix A RT Reaction for USA Customers Using ReactionReady First Strand cDNA Synthesis Kit Catalog Number C 01 Note for customers outside the USA For your convenience an alternative RT Reaction protocol using the recommended components from other manufacturers See the Materials Provided section is described in Appendix B 2 RT Reaction a Prepare the Annealing Mixture For each RNA sample combine the following into a sterile PCR tube Total RNA 1 0t05 0 pg Buffer P 1 0 ul RNase free H20 to a final volume of 10 0 ul Mix the contents gently with a pipettor followed by brief centrifugation Place the mixture in a thermal cycler at 70 C for 3 min Cool to 37 C and keep at that temperature for 10 min b Prepare the RT Cocktail This mixture can be prepared while the Annealing Mixture is incubating at 37 C RT Cocktail 1 reaction 2 reactions 4 reactions 8 reactions Buffer BC 5X RT Buffer 4 ul 8 ul 16 ul 32 ul RNase free H20 4 ul 8 ul 16 ul 32 ul RI RNase Inhibitor 1 ul 2 ul 4 ul 8 ul RE Reverse Transcriptase 1 ul 2 ul 4 ul 8 ul Warm the RT Cocktail at 37 C for 1 min before proceeding to the next step c RT Reaction Add 10 ul of RT Cocktail to each 10 ul Annealing Mixture Mix well but gently with a pipetto
10. hould include the following information Caller s contact information Product name catalog number and quantity Purchase order number or credit card information Visa or MasterCard Shipping address Billing address For more information visit us at http www superarray com SuperArray Bioscience Corp 7320 Executive Way Suite 101 Frederick MD 21704 USA 2 SuperArray MultiGene 12 RT PCR Profiling Kit User Manual Version 1 1 8 15 2003 3 TABLE OF CONTENTS l Background and Introduction 4 ll Kit Contents Materials Provided 6 lll Additional Materials Required 6 IV Protocol 7 Appendix A RT Reaction for USA Customers 9 Appendix B RT Reaction for International Customers 10 V Troubleshooting Guide 11 LIMITED PRODUCT WARRANTY This product is intended for research purposes only and is not intended for drug or diagnostic purposes or for human use This warranty limits our liability to replace this product in the event the product fails to perform due to any manufacturing defect SuperArray Bioscience Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose SuperArray Bioscience Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product NOTICE TO PURCHASER The purchase of MultiGene 12 RT PCR
11. ic primers for several genes with the same annealing temperature can be difficult tedious time consuming and expensive Taking advantage of our optimized proprietary gene specific primers and our long in house gene list from producing pathway specific gene expression arrays we have developed the MultiGene 12 RT PCR Profiling Kits that allow researchers to determine the relative expression of eleven genes in up to eight experimental samples at once The primers and reagents have been formatted in a way to make the kit easy to use economical and accessible for routine use in every research laboratory Each MultiGene 12 RT PCR Profiling Kit contains eight identical strips of 12 PCR tubes Each tube in a strip contains a forward and reverse primer set for a specific gene The strip represents a very focused set of eleven closely related genes from a specific biological pathway The twelfth tube contains primers for a housekeeping gene to control for RNA loading The primers have been immobilized on the bottom of the tubes to allow ambient temperature shipping and easy re suspension into PCR buffer The primers are visible as an orange spot on the bottom of the tube Eight tubes of a pre formulated master mix for PCR are also included in the kit as a lyophilized powder again for ambient temperature shipping To complete the RT PCR analysis experimental RNA samples must also be converted to first strand cDNA the template for the polymerase cha
12. in reaction We offer a kit sold separately Catalog Number C 01 for this step of this procedure Features of the MultiGene 12 RT PCR Profiling Kit Profiles eleven closely related genes in a single analysis Processes up to eight different experiments at once Contains carefully designed and tested gene specific primer sets Provides a simple way to multiplex your RT PCR analyses SuperArray MultiGene 12 RT PCR Profiling Kit User Manual Version 1 1 8 15 2003 Overview of MultiGene 12 RT PCR Profiling Kit Procedure ReactionReady PCR master mix 1000 P 1000A Forward and Reverse Primer Sets for Individual Genes Experimental RNA aliquot 000000000 gt XX 1 2 COQOCOOCOOO00O0 2 CODCOD OOOD0D000 4 gt COQOO00000000 gt X 5 D 000000000000 a a COCOOOOOONGO zi 000000000000 0000000000 C 01 37 C 60 min PH nnn or PM nnn ReactionReady First Strand cDNA Synthesis Kit PCR lt 30 min Product Characterization Agarose Gel Electrophoresis 40 min Image Acquisition And Data Analysis lt 30 min SuperArray MultiGene 12 RT PCR Profiling Kit User Manual Version 1 1 8 15 2003 6 ll Material Provided ReactionReady PCR master mix 1000 P 1000A for end point analysis Eight tubes containing enough lyophilized PCR master mix for 12 reactions each MutliGene 12 Primer Strips Eight strips of 12 PCR tubes each tube containing a set of immobilized forward and
13. incubating at 37 C Dilute 100 mM stock 1 40 with RNase free H20 RT Cocktail 1 reaction 2 reactions 4 reactions 8 reactions 5X Promega Reaction Buffer 4 ul 8 ul 16 ul 32 ql 2 5 mM dNTP Mix 4 ul 8 ul 16 ul 32 ul RNase Inhibitor 1 ul 2 ul 4 ul 8 ul MMLV Reverse Transcriptase 1 ul 2 ul 4 ul 8 ul Warm the RT Cocktail at 37 C for 1 min before proceeding to the next step c RT Reaction Add 10 ul of RT Cocktail to each 10 ul Annealing Mixture Mix well but gently with a pipettor and continue incubation at 37 C for 60 min Heat at 95 C for 5 min to hydrolyze the RNA and to inactivate the reverse transcriptase Hold the finished RT Reaction on ice until the next step Continue the MultiGene 12 Protocol from Step 3 MultiGene 12 PCR page 7 SuperArray MultiGene 12 RT PCR Profiling Kit User Manual Version 1 1 8 15 2003 11 V TROUBLESHOOTING GUIDE amp FREQUENTLY ASKED QUESTIONS Why are the sizes of my PCR products not the same as the expected size listed on the product sheet Why do see multiple bands in the RT PCR product We test each primer set using several sources of RNA and using cDNA libraries Under these conditions the RT PCR product is a single band with the predicted and expected size However your RNA experimental sample may contain alternative splicing variants of a particular message Each variant may produce a band of a different size in the RT PCR reaction depen
14. n A 458 ta M8787 NM_010735 12 GAPDH 419 _ Gapd________ Mm 5289 NM_008084 The actual size of the observed bands may vary or multiple bands may appear in the same sample if splice variants of a particular message exist in your experimental RNA sample Related Products from SuperArray Bioscience 1 ReactionReady First Strand cDNA Synthesis Kit C 01 2 GEArray Q Series Mouse Cytokine and Inflammatory Response Arrays 3 MultiGene 12 Mouse Cytokine I RT PCR Profiling Kit 4 SureSilencing siRNA Kits N SuperArray Bioscience Corporation MultiGene 12 RT PCR Profiling Kit PATHWAY SPECIFIC RT PCR GENE EXPRESSION PROFILING SYSTEMS User Manual SuperArray Bioscience Co 7320 Executive Way Suite 101 Frederick MD 21704 Telephone 301 682 9200 1 888 503 3187 Fax 301 682 7300 1 888 465 9859 Web site www superarray com FOR RESEARCH USE ONLY Version 1 1 8 15 2003 SuperArray MultiGene 12 RT PCR Profiling Kit User Manual Version 1 1 8 15 2003 A SuperArray Bioscience Corporation MultiGene 12 RT PCR Profiling Kit PATHWAY SPECIFIC RT PCR GENE EXPRESSION PROFILING SYSTEMS USER MANUAL ORDERING INFORMATION AND TECHNICAL SERVICE e TEL 1 888 503 3187 301 682 9200 e FAX 1 888 465 9859 301 682 7300 e ON LINE ORDER www superarray com e E MAIL order superarray net customer superarray net support superarray net Orders may be placed by fax e mail or from our website Each order s
15. r and continue incubation at 37 C for 60 min Heat at 95 C for 5 min to hydrolyze the RNA and to inactivate the reverse transcriptase Hold the finished RT Reaction on ice until the next step Continue the MultiGene 12 Protocol from Step 3 MultiGene 12 PCR page 7 SuperArray MultiGene 12 RT PCR Profiling Kit User Manual Version 1 1 8 15 2003 10 Appendix B RT Reaction for Customers Outside the USA Synthesizing first strand cDNA using individual components Individual components for first strand cDNA synthesis reverse transcription may also be purchased separately from other manufacturers See the Materials Provided section of this manual For your convenience the following protocol is provided for using these individual reagents to synthesize first strand cDNA to be used in the MultiGene 12 RT PCR Profiling Kit Customers outside the USA need to buy their components for reverse transcription separately and follow this protocol 2 RT Reaction a Prepare the Annealing Mixture For each total RNA sample combine the following into a sterile PCR tube Total RNA 1 0to5 0 ug Random Primers 1 0 ul RNase free H20 to a final volume of 10 0 ul Mix the contents gently with a pipettor followed by brief centrifugation Place the mixture in a thermal cycler at 70 C for 3 min Cool to 37 C and keep at that temperature for 10 min b Prepare the RT Cocktail This mixture can be prepared while the Annealing Mixture is
16. reverse primers for a specific gene See included product sheet for the specific genes included in this kit lll Additional Materials Required A RNA Isolation Kit See Protocol Section for suggestions B Reverse Transcription RT Reaction OPTION 1 ReactionReady First Strand cDNA Synthesis Kit C 01 See Appendix A OPTION 2 The individual components listed below See Appendix B Reagent Product Kit component Random Primers Promega Catalog C1181 Buffer P RNase Inhibitor Promega Catalog N2511 Component RI Reverse Transcriptase Promega Catalog M1701 Component RE dNTPs Promega Catalog U1330 C 100 bp DNA Step Ladder Promega Catalog Number G695A D The following equipment will also be necessary to use this kit 1 Thermal Cycler 2 Agarose gel electrophoresis equipment and reagents 3 UV Trans Illuminator and high speed or CCD camera SuperArray MultiGene 12 RT PCR Profiling Kit User Manual Version 1 1 8 15 2003 7 IV Protocol 1 Cell Lysis RNA Isolation Total RNA prepared using commercially available kits is suitable for MultiGene 12 RT PCR gene expression profiling analysis Total RNA prepared by Trizol Reagent Invitrogen Life Technologies Cat No 15596 018 RNeasy mini kits Qiagen Cat No 74104 or RNAqueous Ambion Cat No 1911 1912 or 1913 gives good results We use the Qiagen RNeasy mini kits for our QC protocol Total RNA should be dissolved in RNase Free H20 at a concentration of
17. ut different volumes of the RT Reaction For example the scale of the RT Reaction can be doubled and four different MultiGene 12 analyses can be performed adding 20 10 5 or 2 5 ul of RT Reaction to the four different tubes of master mix Examination of these results will quickly reveal the appropriate amount of template needed to achieve the dynamic range of the analysis SuperArray MultiGene 12 RT PCR Profiling Kit User Manual Version 1 1 8 15 2003 12 How should I optimize the number of PCR cycles in my experiment Use a cycle number that produces signal intensities within the dynamic range of the RT PCR method under your experimental conditions amount of input RNA genes being analyzed etc Try not to allow any of the intensities of the gel bands to peak or saturate Otherwise the experimental results will not accurately reflect the relative expression of the genes You may have to optimize the number of cycles for each experiment Lower abundance messages will require more cycles of PCR higher abundance messages fewer cycles Less input RNA may also require more PCR cycles and more RNA fewer cycles In a preliminary experiment you can perform replicate MultiGene 12 analysis for a given RNA sample At the end of a chosen number of cycles remove one of the replicate strips and store it on ice until after the strip using the greatest number of cycles has been removed and you are ready to perform the gel analysis For example
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